JP2009507005A - New ansamycin derivatives - Google Patents

Abstract

A derivative of a benzenoid ansamycin comprising a 1,4-dihydroxyphenyl moiety having an aminocarboxy substituent at the 6-position, wherein the 2-position and the 6-position carboxy substituent are linked by a variable-length aliphatic chain, One or both of the 1-hydroxy and 4-hydroxy positions of the phenyl ring are independently an aminoalkyleneaminocarbonyl group (the alkylene group can optionally be substituted with an alkyl group and have 2 to 3 carbon chains Wherein the derivatized group increases the water solubility and / or bioavailability of the parent molecule but can be removed in vivo. Derivatives are specifically provided. Said compounds are described for the treatment of cancer or B-cell malignancies.
[Selection figure] None

Description

(preface)
The present invention relates to derivatives of ansamycin compounds useful, for example, in the treatment of cancer or B-cell malignancies. In particular, the derivative is a prodrug of an ansamycin compound. The present invention also provides methods for producing these compounds and their use in medicine, particularly in the treatment and / or prevention of cancer or B-cell malignancies.

(Background of the Invention)
The development of highly specific anticancer agents with low toxicity and advantageous pharmacokinetic characteristics is a major challenge in anticancer therapy.
The 90 kDa heat shock protein (Hsp90) is an abundant molecular chaperone that is involved in protein folding and association, many of which are involved in signal transduction systems (reviewed Neckers, 2002; Sreedhar et al., 2004a; Wegele Et al., 2004, and references cited therein.). To date, about 50 of these so-called client proteins have been identified, steroid receptors, non-receptor tyrosine kinases such as the src family, cyclin dependent kinases such as cdk4 and cdk6, cystic transmembrane regulators , Including nitric oxide synthase, and others (Donz and Picard, 1999; McLaughlin et al., 2002; Chiosis et al., 2004; Wegele et al., 2004; http://www.picard.ch/downloads/ Hsp90interactors.pdf). In addition, Hsp90 plays an important role in stress responses and the protection of cells against the effects of mutations (Bagatell and Whitesell, 2004; Chiosis et al., 2004). The function of Hsp90 is complex and involves the formation of a kinetic multienzyme complex (Bohen, 1998; Liu et al., 1999; Young et al., 2001; Takahashi et al., 2003; Sreedhar et al., 2004; Wegele et al., 2004). Hsp90 is a target for inhibitors (Fang et al., 1998; Liu et al., 1999; Blagosklonny, 2002; Neckers, 2003; Takahashi et al., 2003; Beliakoff and Whitesell, 2004; Wegele et al., 2004), resulting in client protein degradation, cell cycle dysregulation, and apoptosis. Recently, Hsp90 has been identified as an important extracellular mediator of tumor invasion (Eustace et al., 2004). Hsp90 has been identified as a therapeutic target for new major cancer therapies and focused detailed studies on Hsp90 function (Blagosklonny et al., 1996; Neckers, 2002; Workman and Kaye, 2002; Beliakoff and Whitesell, 2004; Harris et al. , 2004; Jez et al., 2003; Lee et al., 2004) and the development of high-throughput screening assays (Carreras et al., 2003; Rowlands et al., 2004). Hsp90 inhibitors include compound species such as ansamycin, macrolides, purines, pyrazoles, coumarin antibiotics, and others (reviewed in Bagatell and Whitesell, 2004; Chiosis et al., 2004, and (See cited references.)

Benzenoid ansamycins are a broad class of chemical structures characterized by variable-length aliphatic rings attached to either side of an aromatic ring structure. Natural ansamycins are macbecin and 18,21-dihydromacbecin (also known as macbecin I and macbecin II, respectively) (1 and 2; Tanida et al., 1980 ), Geldanamycin (3; DeBoer et al., 1970; DeBoer and Dietz, 1976; WO 03/106653, and references cited therein), and the herbimycin family (4 5, 6, Omura et al., 1979, Iwai et al., 1980, and Shibata et al., 1986a, WO 03/106653, and references cited therein).

Ansamycins were initially identified as their antibacterial and antiviral activities. However, in recent years, their potential usefulness as anticancer agents has become of great interest (Beliakoff and Whitesell, 2004). A number of Hsp90 inhibitors are currently being evaluated in clinical trials (Csermely and Soti, 2003; Workman, 2003). In particular, geldanamycin has nanomolar utility and apparent specificity for aberrant protein kinases (Chiosis et al., 2003; Workman et al., 2003).
Treatment with Hsp90 inhibitors has been shown to enhance the induction of tumor cell death by radiation and increased cell lethality by combining Hsp90 inhibitors with cytotoxic drugs (e.g., breast cancer, chronic myeloid) Leukemia and non-small cell lung cancer) have also been demonstrated (Neckers, 2002; Beliakoff and Whitesell, 2004). The potential for anti-angiogenic activity is also interesting: Hsp90 client protein HIF-1α plays an important role in the progression of solid tumors (Hur et al., 2002; Workman and Kaye, 2002; Kaur et al. , 2004).
Hsp90 inhibitors also function as immunosuppressants and are involved in complement-induced lysis of several types of tumor cells after Hsp90 inhibition (Sreedhar et al., 2004). Also, treatment with Hsp90 inhibitors results in induced superoxide production (Sreedhar et al., 2004a) associated with immune cell-mediated lysis (Sreedhar et al., 2004). The use of Hsp90 inhibitors as potential antimalarials has also been discussed (Kumar et al., 2003). Furthermore, geldanamycin, composite glycated mammalian prion protein ^{PrP c (complex glycosylated mammalian prion protein} ) has been shown to prevent the formation of (Winklhofer et al., 2003).

  As described above, ansamycin is of interest as a potential anticancer compound and an anti-B-cell malignant compound. However, currently available ansamycins exhibit poor pharmacological or pharmaceutical characteristics. For example, they exhibit poor water solubility, poor metabolic stability, poor bioavailability, or poor pharmaceutical ability (Goetz et al., 2003; Workman paper 2003; Chiosis paper 2004). Herbimycin A and geldanamycin have been identified as inadequate candidates for clinical trials due to their strong hepatotoxicity (reviewed by Workman, 2003), and geldanamycin is the first Excluded from phase clinical trials (Supko et al., 1995, WO 03/106653).

  Geldanamycin is isolated from the culture filtrate of Streptomyces hygroscopicus and exhibits strong activity against protozoa in vitro and weak activity against bacteria and fungi. In 1994, an association of geldanamycin with Hsp90 was shown (Whitesell et al., 1994). The biosynthetic gene cluster of geldanamycin has been cloned and sequenced (Allen and Ritchie, 1994; Rascher et al., 2003; WO 03/106653). The DNA sequence is available under NCBI accession number AY179507. Isolation of a recombinant geldanamycin producing strain derived from S. hygroscopicus subsp. Duamyceticus JCM4427 and 4,5-dihydro-7-O-descarbamoyl- The isolation of 7-hydroxygeldanamycin and 4,5-dihydro-7-O-descarbamoyl-7-hydroxy-17-O-demethylgeldanamycin has recently been described (Hong et al., 2004 ). By supplying geldanamycin to the herbimycin producing strain Streptomyces hygroscopicus AM-3672 (Streptomyces hygroscopicus AM-3672), the compound 15-hydroxygeldanamycin, the tricyclic geldanamycin analog KOSN- 1633 and methyl-geldanamycinate have been isolated (Hu et al., 2004). The two compounds 17-formyl-17-demethoxy-18-O-21-O-dihydrogeldanamycin and 17-hydroxymethyl-17-demethoxygeldanamycin are the herbimycin producing strain Streptomyces hygroscopicus Isolated from S. hygroscopicus NRRL 3602 (S. hygroscopicus NRRL 3602) containing plasmid pKOS279-78 with various genes from AM-3672 (Streptomyces hygroscopicus AM-3672) (Hu et al., 2004) ).

In 1979, the ansamycin antibiotic herbimycin A was isolated from Streptomyces hygroscopicus strain No. AM-3672 and named according to its potent herbicidal effect. The anti-tumor activity was demonstrated by using cells of rat kidney strains infected with temperature-sensitive mutants of Rous sarcoma virus (RSV) for agents that revert the transformed form of these cells ( Review Uehara's paper, 2003.) Herbimycin A is considered to act primarily through binding to the Hsp90 chaperone protein, but its direct binding to the conserved cysteine residue, followed by inactivation of the kinase, has also been discussed. (Uehara's paper, 2003).
A chemical derivative was isolated and a compound with an altered substituent at C19 of its benzoquinone nucleus and a halogenated compound in the answer chain showed lower toxicity and higher antitumor activity than herbimycin A (Omura Et al., 1984; Shibata et al., 1986b). The sequence of the herbimycin biosynthetic gene cluster has been identified in WO 03/106653 and a recent paper (Rascher et al., 2005).

  The ansamycin antibiotics macbecin (1) and 18,21-dihydromacbecin (2) (C-14919E-1 and C-14919E-1) are identified by their antifungal and antiprotozoal activities and are Isolated from the culture supernatant of C-14919 (Nocardia sp No. C-14919) (Antinosynnema pretiosum subsp pretiosum ATCC 31280) (Tanida et al., 1980; Muroi et al. 1980; US 4,315,989 and US 4,187,292). 18,21-Dihydromacbecin is characterized by containing the hydroquinone form of its nucleus. Both macbecin and 18,21-dihydromacbecin have been shown to have similar antibacterial and antitumor activity against cancer cell lines such as the murine leukemia P388 cell line (Ono et al., 1982). . Reverse transcriptase and terminal deoxynucleotidyl transferase activities were not inhibited by macbecin (Ono et al., 1982). The effects of macbecin on Hsp90 inhibitors have been reported in the literature (Bohen, 1998; Liu et al., 1999). After adding macbecin and 18,21-dihydromacbecin to the microbial culture broth, conversion to a compound having a hydroxy group instead of a methoxy group at a specific position or positions is described in U.S. Pat.No. 4,421,687 and U.S. Pat. In the issue.

Antibiotics TAN-420A-E were discovered (7-11, EP 0 110 710) from production strains belonging to the genus Streptomyces at the time of screening of various soil microorganisms.

In 2000, the isolation of geldanamycin-related non-benzoquinone ansamycin metabolite reblastatin from the culture of Streptomyces sp. S6699 and its potential therapeutic value in rheumatoid arthritis was described.
Additional Hsp90 inhibitors, which are different from chemically unrelated benzoquinone ansamycins, are initially due to their antifungal activity, the fungus Monosporium bonorden (see review article Uehara, 2003). It was found to be a 14-membered ring macrolide isolated from Nectria radicicola, which is Radicicol (monorden), which was isolated from Nectria radicicola. It was subsequently identified as an inhibitor of the Hsp90 chaperone protein in addition to its antifungal activity, antibacterial activity, antiprotozoal activity, and cytotoxicity (see review Uehara paper, 2003; Schulte et al paper, 1999). ). The anti-angiogenic activity of radicicol (Hur et al., 2002) and its semisynthetic derivatives (Kurebayashi et al., 2001) has also been described.

  Recent interest has focused on the 17-amino derivatives of geldanamycin (Bagatell and Whitesell, 2004) as new creation of ansamycin anticancer compounds. For example, 17- (allylamino) -17-desmethoxygeldanamycin (17-AAG, 12) (Hostein et al., 2001; Neckers paper, 2002; Nimmanapalli et al., 2003; Vasilevskaya et al., 2003; Smith-Jones et al., 2004), and 17-desmethoxy-17-N, N-dimethylaminoethylamino-geldanamycin (17-DMAG, 13) (Egorin et al., 2002; Jez et al., 2003). More recently, geldanamycin was derivatized at the 17 position to create 17-geldanamycinamide, carbamate, urea, and 17-aryl geldanamycin (Le Brazidec et al., 2003). A library of over 60 17-alkylamino-17-demethoxygeldanamycin analogs has been reported and tested for affinity and water solubility for Hsp90 (Tian et al., 2004). A further approach to reduce the toxicity of geldanamycin is the selective targeting and delivery of active geldanamycin compounds into malignant cells by conjugation to tumor-targeted monoclonal antibodies (Mandler et al., 2000). .

これらの誘導体は、低下した肝毒性を示すが、これらはまだ水溶性にのみ限界があり、可溶化担体の使用が必要とされる(例えば、クレモフォア(Cremophore)(登録商標)、DMSO-卵レシチン)。それ自身が一部の患者において副作用を生じる可能性がある。

These derivatives show reduced hepatotoxicity, but they are still only limited in water solubility and require the use of solubilizing carriers (e.g. Cremophore®, DMSO-egg lecithin) ). It can itself cause side effects in some patients.

  Thus, there remains a need to identify new ansamycins with improved water solubility that would have improved pharmacological and low side effect properties with respect to administration. The present invention is a novel ansamycin that is a prodrug and can be chemically or enzymatically cleaved to its parent molecule and generally has improved pharmaceutical properties compared to currently available ansamycins. Derivatives are disclosed.

(Summary of the Invention)
The present invention provides derivatives of ansamycin, methods for preparing these compounds, intermediates therefor, and methods of using these compounds in medicine. In particular, derivatives of ansamycin are prodrugs.
In its broadest aspect, the present invention provides a derivative of a benzenoid ansamycin comprising a self-cleaving and water-soluble derivatizing group at positions 18 and / or 21 of the parent molecule. These groups are designed to be chemically cleaved to generate a bioactive parent molecule or can be cleaved through enzymatic means.

Accordingly, the present invention provides a benzenoid answer comprising a 1,4-dihydroxyphenyl moiety having an aminocarboxy substituent at the 6-position, wherein the 2-position and 6-position carboxy substituents are linked by a variable-length aliphatic chain. A derivative of mycin, wherein one or both of the 1-hydroxy and 4-hydroxy positions of the phenyl ring are independently substituted by an aminoalkyleneaminocarbonyl group (an alkylene group (optionally an alkyl group such as a methyl group). .) Has a carbon chain length of 2 to 3 and is derivatized by the method, and the derivatized group increases the water solubility and / or bioavailability of the parent molecule but is removed in vivo. It relates to said derivative, characterized in that it is possible.
In this context, “parent molecule” means the corresponding molecule having underivatized hydroxyl groups at positions 1 and 4 of the phenyl ring (ie hydroquinone) or its benzoquinone form.

（式中、
R₁はH、OH、OMe、-NHCH₂CH=CH₂、又は-NHCH₂CH₂N(CH₃)₂を表し；
R₂はOH、又はケトを表し；
R₃はOH、又はOMeを表し；
R₅はH、又は

（式中、
nは0又は1を表し；
R₆はH、Me、Et、又はイソ-プロピルを表し；
R₇、R₈、及びR₉は、それぞれ独立に、H、又はC1-C4分枝鎖若しくは直鎖アルキル基を表すか；或いはR₇とR₈、又はR₈とR₉が6員炭素環を形成するように結合することができ；
R₁₀はH、又はC1-C4分枝鎖若しくは直鎖アルキル基を表す。）を表し
；
但し、R₅部位が両方ともHではなく、かつR₅のどちらもがHではない場合、2つのR₅部位は同一であることを条件とする。）。
上記構造は代表的な互変異性体を示し、かつ本発明は式(IA)、(IB)、及び(IC)の化合物の全ての互変異性体（例えば、エノール化合物が示される場合のケト化合物、及び逆もまた同様）を包含する。
式(IA)、(IB)、及び(IC)の化合物は、上記において、まとめて式(I)の化合物と呼ぶ。
更なる態様において、本発明は、医薬として使用するための、式(I)の化合物などのアンサマイシン誘導体、又はその医薬として許容し得る塩を提供する。 In a further particular embodiment, the present invention provides an ansamycin derivative of formula (IA-IC): or a pharmaceutically acceptable salt thereof:

(Where
R ₁ represents H, OH, OMe, —NHCH ₂ CH═CH ₂ , or —NHCH ₂ CH ₂ N (CH ₃ ) ₂ ;
R ₂ represents OH or keto;
R ₃ represents OH or OMe;
R ₅ is H, or

(Where
n represents 0 or 1;
R ₆ represents H, Me, Et, or iso-propyl;
R ₇ , R ₈ , and R ₉ each independently represent H, or a C1-C4 branched or straight chain alkyl group; or R ₇ and R ₈ , or R ₈ and R ₉ are 6-membered carbons Can be linked to form a ring;
R ₁₀ represents H, or a C1-C4 branched chain or straight chain alkyl group. );
However, if both R ₅ sites are not H and neither R ₅ is H, the two R ₅ sites are the same. ).
The above structures represent representative tautomers and the present invention covers all tautomers of compounds of formulas (IA), (IB), and (IC) (eg, keto when enol compounds are shown). Compounds, and vice versa).
Compounds of formula (IA), (IB), and (IC) are collectively referred to above as compounds of formula (I).
In a further aspect, the present invention provides an ansamycin derivative, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof for use as a medicament.

(Definition)
The articles “a” and “an” mean one or more (ie, at least one) of the grammatical objects of the article. By way of example, “an analogue” means one analogue or more than one analogue.
As used herein, the term “analog” is a compound that is structurally similar to another but slightly different in composition (substitution of one atom by another, or the presence of a particular functional group or Non-existent).

  As used herein, the term “cancer” arises from epithelial tissue, is found in the skin, or more commonly the inner surface of a body organ, such as the breast, prostate, lung, kidney, pancreas, stomach, or intestine. Means malignant neoplasm. Cancer tends to penetrate nearby tissues and spread (metastasize) to distant organs, such as bone, liver, or brain. As used herein, the term “cancer” includes, but is not limited to, metastatic tumor cell types such as melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma, and, without limitation, the colorectal Includes both types of tissue carcinomas such as cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, kidney cancer, gastric cancer, glioblastoma, primary liver cancer, and ovarian cancer.

  The term “bioavailability” as used herein refers to the degree or rate at which a drug or other substance becomes absorbed or available at a bioactive site after administration. This property depends on many factors such as the solubility of the compound, the rate of absorption in the intestine, the extent of protein binding, and metabolism. Various tests for bioavailability are known to those skilled in the art and are described herein (see also Egorin et al. (2002)).

  The term “B-cell malignancy” as used herein encompasses a group of disorders including chronic lymphocytic leukemia (CLL), multiple myeloma, and non-Hodgkin lymphoma (NHL). These are neoplastic diseases of the blood and hematopoietic organs. These cause bone marrow dysfunction and immune system dysfunction and the host is highly sensitive to infection and bleeding.

  The term “prodrug” as used in this application means a precursor or derivative form of a pharmaceutically active substance that has improved formulation properties compared to the original drug. It can be, for example, less cytotoxic or highly soluble than the original drug and can be activated (eg, chemically or enzymatically cleaved) or more effective. (E.g. Wilman DEV (1986) Pro-drugs in Cancer Chemotherapy) Biochemical Society Transactions 14, 375-382 (615th Meeting, Belfast), and Stella VJ et al. (1985) "Prodrugs: A Chemical Approach to Targeted Drug Delivery" Directed Drug Delivery R. Borchardt et al. (Edited), pages 247-267 ( See Humana Press).

The term “water-soluble” as used in this application refers to solubility in aqueous media such as phosphate buffered saline (PBS) pH 7.4, or 5% glucose solution. The water solubility test is shown in the examples below as a “water solubility assay”.
As used herein, the term “ansamycin derivative” means a benzenoid ansamycin derivative. In its broadest aspect, it represents the above representative of the present invention. For example, the compound of the above formula (I) or a pharmaceutically acceptable salt thereof. These compounds are also referred to as “compounds of the invention” or “derivatives of ansamycin” and these terms are used interchangeably in this application.

  Pharmaceutically acceptable salts of the compounds of the invention, such as the compounds of formula (I), include pharmaceutically acceptable inorganic acids or bases, or conventional salts formed from organic acids or bases, as well as quaternary ammonium acid additions. Contains salt. Specific examples of suitable acid salts include hydrochloride, hydrobromide, sulfate, phosphate, nitrate, perchlorate, fumarate, propionate, succinate, glycolate , Formate, lactate, maleate, tartrate, citrate, palmoic acid, malonate, hydroxymaleate, phenylacetate, glutamate, benzoate, salicylate, fumaric acid Salt, toluenesulfonate, methanesulfonate, naphthalene-2-sulfonate, benzenesulfonate, hydroxynaphthoate, hydroiodide, malate, tannate, and the like. Of particular interest is the hydrochloride salt. Other acids such as oxalic acid are not pharmaceutically acceptable per se, but may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts. Specific examples of suitable base salts include sodium salt, lithium salt, potassium salt, magnesium salt, aluminum salt, calcium salt, zinc salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, choline salt, There are diethanolamine salts, ethylenediamine salts, N-methylglucamine salts, and procaine salts. The following references to the compounds of the present invention include both compounds of formula (I) and their pharmaceutically acceptable salts.

Alkyl, alkenyl, and alkynyl groups may be straight or branched.
Examples of alkyl, for example C1-C4 alkyl groups, are methyl, ethyl, n-propyl, i-propyl, and n-butyl.
As used herein, the term “18,21-dihydromacbecin” and macbecin II (the hydroquinone of macbecin I) are used interchangeably.

(Description of the present invention)
Strategies to improve the physical properties of drug candidates (e.g., bioavailability) are usually prodrug precursors that depend on environmental effects such as enzymatic hydrolysis to release the active original drug Is to use the body. However, due to changes between individuals, the release of the required amount of active agent in vivo may not be achieved.

  The present invention provides derivatives of ansamycin having another method of controlling the rate of active agent release. This approach takes advantage of the self-cleavage of the incorporated amino side chain that occurs at physiological pH via an intramolecular cyclization elimination reaction. Said intramolecular attack on the carbamate functionality by the terminal amino group yields a cyclic urea fragment, resulting in the release of the original drug.

The rate of drug release depends on the chemical cyclization rate constant and is more related to substituents than external effects.
Although it is contemplated that the compounds of the present invention may undergo chemically mediated self-cleavage, they can also be substrates for enzymatic cleavage, and are within the scope of the present invention. Is done.

Accordingly, the present invention provides derivatives of ansamycin, methods for producing these compounds, intermediates therefor, and methods of using these compounds in medicine, as indicated above.
In examples of a series of compounds of formula IA-IC, R ₆ represents H, Me, or Et. In a further example of the series of compounds of formula IA-IC, R ₁₀ represents a C1-4 branched or linear alkyl group. In a further example of the series of compounds of formula IA-IC, R ₆ represents H, Me, or Et and R ₁₀ represents a C 1-4 branched or linear alkyl group.

Each of the R ₅ groups is the same or H and the other is substituted as described herein. In one example embodiment, one of the R ₅ groups is H; in another example embodiment, neither of R ₅ is H. Preferably, the R ₅ group of C21 is H.
Preferably R ₆ represents Me. Alternatively, preferably R ₆ represents Et.
Preferably R ₁₀ represents Me. Alternatively, preferably R ₁₀ represents Et.

Preferably R ₇ represents H. Preferably R ₈ represents H. Preferably R ₉ represents H. When R ₇ and R ₈ or R ₈ and R ₉ are joined so as to form a 6-membered carbocycle, the ring may suitably be a cyclohexyl ring.
Preferably n = 0.
With respect to the ansamycin ring, the side chain stereochemistry preferably follows that of the corresponding parent compound (ie, macbecin, geldanamycin, herbimycin A, see for example the structures shown in Table 4 above).

The compound of formula (I) can represent, for example, derivatives of the following compounds:
・ Macbecin (Formula (IA));
Geldanamycin (formula (IB), wherein R ₁ represents OMe, particularly when R ₂ represents OH);
Herbimycin B (Formula (IB), wherein R ₁ represents H, particularly when R ₂ represents OH);
· 17-AAG (formula (IB) (In the formula, R _1, especially when R ₂ represents OH, represents a _{NHCH 2 CH = CH 2).} ;
17-DMAG (formula (IB) (wherein R ₁ represents NHCH ₂ CH ₂ NMe ₂ particularly when R ₂ represents OH));
Herbimycin A (Formula (IC) (wherein R ₂ represents OMe and R ₃ represents OMe); or Herbimycin C (Formula (IC) (wherein R ₂ represents OH) And R ₃ represents OMe).

（式中、Lは脱離基である。）、
式(H)の化合物

（式中、Pは保護基を表す。）
と反応させることにより、R₅部位のどちらもがHではない式(I)の化合物を調製すること：又は
(b) 式(IID)、(IIE)、若しくは(IIF)の化合物、又はその保護誘導体を

（式中、Lは脱離基である。）、
式(H)の化合物

（式中、Pは保護基を表す。）
と反応させることにより、C21のR₅部位がHである式(I)の化合物を調製すること；又は
(c) 式(I)の化合物又はその塩を、式(I)の別の化合物又は別のその医薬として許容し得る塩に変換すること；又は
(d) 式(I)の保護化合物を脱保護することを含む。
上記文章において、式(IIA)、(IIB)、(IIC)、(IID)、(IIE)、及び(IIF)の化合物は、まとめて式(II)の化合物と呼ぶ。 In general, the compounds of the present invention are prepared by semi-synthetic derivatives of a group of ansamycin compounds.
Accordingly, a process for preparing a compound of formula (I), or a pharmaceutically acceptable salt thereof, is:
(a) a compound of formula (IIA), (IIB), or (IIC), or a protected derivative thereof

(Wherein L is a leaving group),
Compound of formula (H)

(In the formula, P represents a protecting group.)
Preparing a compound of formula (I) in which neither of the R ₅ sites is H by reaction with: or
(b) a compound of formula (IID), (IIE), or (IIF) or a protected derivative thereof

(Wherein L is a leaving group),
Compound of formula (H)

(In the formula, P represents a protecting group.)
By reaction with, it R ₅ sites C21 to prepare compounds of formula (I) is H; or
(c) converting a compound of formula (I) or a salt thereof into another compound of formula (I) or another pharmaceutically acceptable salt thereof; or
(d) deprotecting the protected compound of formula (I).
In the above text, the compounds of formula (IIA), (IIB), (IIC), (IID), (IIE), and (IIF) are collectively referred to as compounds of formula (II).

In steps (a) and (b), exemplary leaving groups L are halogen (e.g., chlorine, bromine), alkoxy (e.g., C1-4 alkoxy), aryl (e.g., phenoxy or 4-nitrophenoxy, etc.). Substituted phenoxy), or alkylaryl (eg, C1-4 alkylaryl, eg, benzyloxy). Preferably L represents 4-nitrophenoxy. Exemplary protecting groups P include t-butyloxycarbonyl ("Boc") and trityl groups.
The reaction of the compound of formula (II) with the compound of formula (H) can be carried out under conventional conditions known per se for carbamate formation. For example, refluxing the component in an inert solvent such as dichloromethane, post-treatment with water.

式(J)の化合物：

（式中、L'は脱離基を表し、好ましくはその1つはLよりも不安定である。）
と反応させることにより、製造することができる。例証的なL'基は、上記のLに関して記載されているものである。式Jの好ましい化合物は、4-ニトロフェニルクロロホルマートである。 The compound of formula (II), or a protected derivative thereof, is a compound of formula IIIA-IIIC, or a protected derivative thereof:

Compound of formula (J):

(Wherein L ′ represents a leaving group, preferably one of which is more unstable than L.)
It can manufacture by making it react. Exemplary L ′ groups are those described for L above. A preferred compound of formula J is 4-nitrophenyl chloroformate.

The reaction of the compound of formula (III) with the compound of formula (J) can be carried out under conventional conditions known per se, for example, refluxing of the component in an inert solvent such as dichloromethane.
Since the OH group of C18 is more reactive than the OH group of C21, the compounds of formulas (IID) to (IIF) can be converted to the corresponding compounds of formulas (IIIA) to (IIIC) with a slight excess of formula J It can obtain by making it react with the compound of this. Compounds of formula (IIA)-(IIC) can be obtained by reacting the corresponding compounds of formula (IIIA)-(IIIC) with more than a 2-fold excess of compound of formula J.

Compounds of formula IIIA-IIIC ("compound of formula (III)" below) and protected derivatives thereof can be prepared as follows:
Initially, native ansamycin for use as a template can be obtained via direct fermentation of a strain producing the desired compound. One skilled in the art will be able to culture the production strain under conditions suitable for the production and isolation of natural product templates. The strains shown in Table 1 are examples of production strains for natural product templates. One skilled in the art will recognize that there are other strains available that will produce the same compound under appropriate conditions.

Alternatively, the compounds may be commercially available: Table 2 shows the compounds that can be purchased, along with their catalog numbers.

  In addition to the specific methods and references cited herein, one of ordinary skill in the art, but not limited to, “Vogel's Textbook of Practical Organic Chemistry” (Furniss et al., 1989), and standard textbooks for synthetic methods such as “March's Advanced Organic Chemistry” (Smith and March, 2001).

Natural ansamycin exists and can be isolated primarily in benzoquinone form. In some cases, the hydroquinone form can be isolated from the fermentation broth. If the benzoquinone form is isolated, it will need to be converted to the corresponding compound of formula (III) (hydroquinone). It is well known that benzoquinone can be chemically converted (reduced) to hydroquinone and vice versa (oxidized). This can be applied to the above ansamycin natural products so that hydroquinone can be synthesized by various methods when the benzoquinone form occurs in nature. By way of example (but not by way of limitation), this can be achieved in an organic medium using a hydride source such as but not limited to LiAlH ₄ or SnCl ₂ —HCl. Alternatively, this transformation involves dissolving the benzoquinone form of ansamycin in an organic medium followed by an aqueous solution of a reducing agent such as but not limited to sodium hydrosulfite (Na ₂ S ₂ O ₄ or sodium thionite). Can be mediated by washing with. Preferably, this transformation is performed by dissolving macbecin or geldanamycin in ethyl acetate and mixing the solution vigorously with aqueous sodium hydrosulfite (Muroi et al., 1980). The resulting organic solution is then washed with water, dried, and the solvent removed under reduced pressure to give approximately quantitative amounts of 18,21-dihydromacbecin or 18,21-dihydrogeldana, respectively. Mycin can be obtained.

  Compounds of formula (IIB) and (IIC) such as those derived from geldanamycin may or will contain a secondary hydroxyl group at the C-11 position. In order to derivatize these compounds with C-18 hydroxyl, it may be necessary exclusively to first modify the C-11 hydroxyl. Furthermore, compounds derivatized at the C-11 position together with derivatization at C-18 or C-21 are clearly considered as compounds of the invention in their possession. The following is a method for achieving that of geldanamycin, but those skilled in the art will recognize that other compounds of formula (IIB) and (IIC), (IIE) and (IIF) where the parent compound is C-11 and contain an OH group. It will be appreciated that these can be applied to compounds as well.

  For example, the C-11 hydroxyl includes, but is not limited to, swannated, Dess-Martin periodination, tetrapropylammonium peruruthenate (TPAP), Jones reagent, pyridinium chlorochromate (Corley's reagent ( Corey's reagent)), or can be oxidized to ketones by one of many standard protocols for oxidizing secondary alcohols such as pyridinium dichromate to ketones. Using 11-oxogeldanamycin, the benzoquinone can be immediately reduced to hydroquinone as described above. One skilled in the art will recognize that care must be taken not to reduce newly formed C-11 ketones simultaneously. In other words, the reducing agent selected should be chemoselective for the benzoquinone system over the C-11 carbonyl. Such chemoselective materials are known to those skilled in the art, an example of a suitable material (without limitation) is sodium hydrosulfite, and an inappropriate material includes, but is not limited to, LiAlH4. 11-oxo-18,21-dihydrogeldanamycin can then be used as a template for further derivatization into compounds of formula I as described below.

As will be appreciated by those skilled in the art, protecting groups can generally be used in the synthesis of the compounds and intermediate compounds of the invention if desired.
The hydroquinone ansamycins represented by formula (III) can be converted from their benzoquinone form, if necessary, and used as a template for further semisynthesis (ie, step (a)).

Other compounds encompassed by the present invention can be prepared by methods described herein and / or methods known to those skilled in the art.
Compounds of formula (H) can be prepared by methods known to those skilled in the art. One suitable method for preparing the compound of formula H is shown below, but it will be appreciated that other suitable methods can be used as well.

  Thus, in one embodiment, various self-cleaving and water-soluble side chains can be introduced at positions 18 and / or 21 of 18,21-dihydromacbecin. This treats mono-N-protected diamines of variable long chain and substitution pattern (H) with an intermediate, a 4-nitrophenyl carbonate analog of macbecin (eg, compound of formula (II) above). Can be achieved.

A primary aminoalkyl benzyl ether (A) derived from manipulation of a protecting group on an amino alcohol is reacted with a benzaldehyde to give a Schiff base (B). The formed imine is treated with an alkylating agent such as trialkyloxonium tetrafluoroborate to introduce R ₁₀ to give the iminium salt (C). Hydride reduction to benzylamine (D) followed by di-debenzylation via catalytic hydrogenolysis provides secondary amines of type (E). N-Boc protection to (F) is followed by hydroxyl activation to a suitable leaving group such as mesylate (G). If necessary, the Boc group of the compounds (F), (G), and (H) in the figure can be replaced with another protecting group by treating the compound (E) with another protecting reagent. it can. Another preferred protecting group is trityl.

  In step (c), salt formation and conversion can be performed by conventional methods known to those skilled in the art. The interconversion of the compound of formula (I) can be performed by a known process. For example, hydroxy and keto groups can be interconverted by oxidation / reduction as described elsewhere herein.

  In steps (a), (b), and (d), examples of protecting groups and methods for their removal are described in TW Greene “Protective Groups in Organic Synthesis” (J Wiley and Sons, 1991 ) Can be found. Suitable hydroxyl protecting groups are alkyl (e.g. methyl), acetal (e.g. acetonide), and acyl (e.g. acetyl or benzoyl) that can be removed by hydrolysis, and arylalkyl that can be removed by catalytic hydrogenolysis. (Eg benzyl) or silyl ethers that can be removed by acidic hydrolysis or fluoride ion assisted cleavage. Suitable amine protecting groups include sulfonyl (e.g. tosyl), acyl (e.g. benzyloxycarbonyl or t-butoxycarbonyl), and arylalkyl (e.g. benzyl) which can be removed by hydrolysis or hydrogenolysis as required. Including.

(式中、R₁、R₂、R₃、及びR₅は、上記で定義したもの(R₅がHを表さないことを除く)であり、かつPaは保護基を表す。)。例証的なヒドロキシル保護基Paは、上記のもの、特にシリルエーテルを含む。そのシリル基の除去による脱保護は、従来の条件下での加水分解により達成することができる。 Compounds of formula (I) in which C18 R ₅ is H can be prepared by deprotection of compounds of formula (IVA)-(IVC), or further protected derivatives thereof (collectively `` formula (IV) Known as “a compound of”):

(Wherein R ₁ , R ₂ , R ₃ , and R ₅ are those defined above (except that R ₅ does not represent H), and Pa represents a protecting group.) Exemplary hydroxyl protecting groups Pa include those described above, particularly silyl ethers. Deprotection by removal of the silyl group can be achieved by hydrolysis under conventional conditions.

（式中、Ｌは脱離基である。）、
式Hの化合物と反応させることにより製造することができる。
適切な条件は、工程(a)及び(b)に関して上記で与えられたものを含む。 Compounds of formula (IV), or further protected derivatives thereof, are compounds of formula (VA)-(VC), or protected derivatives thereof (collectively known as “compounds of formula (V)”):

(Wherein L is a leaving group),
It can be prepared by reacting with a compound of formula H.
Suitable conditions include those given above for steps (a) and (b).

式(J)の化合物と反応させることにより製造することができる。
適切な条件は、式(II)化合物の式(J)化合物との反応に関して上記で与えられたものを含む。 Compounds of formula (V), or protected derivatives thereof, are compounds of formulas (VIA)-(VIC), or protected derivatives thereof (collectively known as “compounds of formula (VI)”):

It can be produced by reacting with a compound of formula (J).
Suitable conditions include those given above for the reaction of a compound of formula (II) with a compound of formula (J).

  The compound of formula (VI) or a protected derivative thereof can be produced by protecting the compound of formula (III). When Pa represents a silyl ether, protection can be achieved by treating the compound of formula (III) with trialkylsilyl chloride or trialkylsilyl triflate in the presence of a base.

Other compounds of the invention can be prepared by methods known per se or by methods analogous to those described above.
The compounds of the present invention are useful as templates for direct and further semisynthetic or biotransformation, producing compounds useful as anticancer agents. Methods for semi-synthetic derivatization of ansamycins such as geldanamycin and related compounds are well known in the art and include, for example, the modification methods described in WO 03/013430, WO 02/079167, WO 03/066005 (But not limited.)

In particular, the compounds of the present invention have utility as prodrugs, which can be chemically cleaved to produce the active parent compound. Cleavage assays for assessing the cleavage rate are known in the art.
The above structure of the intermediate may tautomerize and if a representative tautomer is indicated, it and all tautomers will be understood. For example, when an enol compound is indicated, it is intended to refer to a keto compound, and vice versa.

In addition, novel compounds of formulas (II), (III), (IV), (V), and (VI) and protected derivatives thereof are claimed as embodiments of the present invention.
The present invention further provides for the use of the compounds of the present invention in the treatment of cancer or B-cell malignancies. Also provided are compounds of the invention for use in the treatment of cancer or B-cell malignancies. Also provided is a method of treating cancer or a B-cell malignancy comprising administering to a patient an effective amount of a compound of the present invention. Also provided is the use of a compound of the invention in the manufacture of a medicament for the treatment of cancer or B-cell malignancies.

  Ansamycin is not limited, but treatment of heart failure and stroke (US 6,174,875, WO 99/51223), treatment of fibrosis (WO 02/02123), treatment or prevention of restenosis (WO 03/079936), Treatment or prevention of diseases associated with protein aggregation and amyloid function (WO 02/094259), treatment of peripheral nerve damage and promotion of nerve regeneration (WO 01/03692, US 6,641,810, EP 1 024 806, US 2002/0086015, US 6,210,974, WO 99/21552, US 5,968,921), and inhibition of angiogenesis (WO 04/000307) are known to have utility in the treatment of other conditions. Also, uses and methods involving the compounds of the present invention extend to these other indications.

In a preferred embodiment, the present invention provides compounds that have utility in the treatment of cancer. One skilled in the art will be able to determine the ability of these compounds to inhibit tumor cell growth by monotonous experiments (Tian et al., 2004; Hu et al. 2004; Dengler et al., 1995). Please refer to it.)
The present invention also provides a pharmaceutical composition comprising an ansamycin derivative or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

  Existing ansamycin HSP90 inhibitors such as geldanamycin, 17-AAG, and 17-DMAG have or have poor physiological properties, poor water solubility, and poor bioavailability in clinical trials. Yes. The present invention provides an ansamycin derivative having improved properties such as solubility and / or bioavailability. One skilled in the art can readily determine the solubility of a given compound of the invention using standard methods. Exemplary methods are given in the examples herein.

  In addition, one of ordinary skill in the art will be able to use the in vivo and in vitro methods known to those skilled in the art such as, but not limited to, those described below and in the paper of Egorin MJ et al. The availability of physics can be measured. The bioavailability of a compound is measured by various factors (e.g., water solubility, intestinal absorption rate, range of protein binding, and metabolism), each of which can be measured by an in vitro test, as described below. . It will be appreciated by those skilled in the art that an improvement of one or more of these factors will lead to an improvement in the bioavailability of the compound. The bioavailability of a compound can also be measured using in vivo methods as described in more detail below.

(In vitro assay)
a) Caco-2 permeation assay
Confluent Caco-2 cells in a 24-well Corning Costar Transwell configuration (Li, AP, 1992; Grass, GM et al., 1992, Volpe, DA et al., 2001) and described herein To determine the permeability and efflux rate of the compound. Suitable configurations include those provided by In Vitro Technologies Inc. Baltimore, Maryland, USA. In a suitable configuration, the top chamber contains 0.15 mL HBBS pH 7.4, 1% DMSO, 0.1 mM Lucifer Yellow, and the bottom chamber contains 0.6 mL HBBS pH 7.4, 1% DMSO. Control and test assays are incubated at 37 ° C. with shaking at 130 rpm in a humidified incubator. Lucifer Yellow can permeate only through the paracellular pathway (i.e., between tight junctions), and for Lucifer Yellow, high Apparent Permeability (P _app ) reduces cell damage between assays. All such wells shown and discarded. In addition to the parent compound, suitable reference controls include propranolol with good passive permeability without known transport effects and acebutalol with poor passive permeability attenuated by active efflux by P-glycoprotein.

流出比の正の値は、細胞の頂端面から活性な流出を示す。従って、改善された生物学的利用能は、その親分子と比べて、本発明の化合物に関して増加したP_app、及び/又は低下した流出比により、上記アッセイにおいて示される。 Compounds are tested (at 0.01 mM) by applying the compound in the top or bottom chamber, in unilateral and bidirectional configurations. Compounds in the top or bottom chamber are analyzed by LC-MS. The results are expressed as the apparent transmittance P _app , (nm / s) and as the outflow ratio (A to B vs B to A).

A positive value for the efflux ratio indicates an active efflux from the apical surface of the cell. Thus, improved bioavailability is demonstrated in the above assay by an increased P _app and / or a decreased efflux ratio for the compound of the invention relative to its parent molecule.

b) Stability assay for human liver microsomes (HLM) Increased metabolic stability is also associated with improved bioavailability, which can be measured, for example, using the HLM assay as shown below. it can. Liver homogenates provide a measure of compound-specific vulnerability to first-phase (oxidation) enzymes such as CYP450s (eg CYP2C8, CYP2D6, CYP1A, CYP3A4, CYP2E1), esterases, amidases, and flavin monooxygenases (FMOs).

The half-life (T1 / 2) of compounds can be measured by monitoring their disappearance over a long period of time by LC-MS in the state exposed to human liver microsomes. 0.001 mM compound, 40 min at 37 ° C, 0.1 M Tris-HCl, pH 7.4, with a sub-cellular fraction of liver human microsomes at 0.25 mg / mL protein and saturating NADPH as a cofactor Incubate with. At specified time intervals, acetonitrile is added to the test sample to precipitate protein and stop metabolism. Samples are centrifuged and analyzed for parent compound.
Improved bioavailability is shown in the above assay by an increased T1 / 2 compared to the parent compound.

(In vivo assay)
In vivo assays can also be used to determine the bioavailability of a compound. In general, compounds are administered to test animals (e.g., mice or rats) both intraperitoneally (ip) or intravenously (iv), and orally (po), and blood samples are taken at regular intervals for extended periods of time. Over time, investigate how much the plasma concentration of the drug fluctuated. Using the time course of plasma concentration over time, the absolute bioavailability of the compound can be calculated as a percentage using a standard model. Examples of typical protocols are described below. Mice are administered ip, iv, or po with a compound of the invention or parent compound 1, 10, or 75 mg / kg. Blood samples are taken at 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, 420, and 2880 minutes, and the concentration of the compound of the invention in the sample is determined via HPLC taking measurement. The plasma concentration over time is then used to determine the important parameters, the area under the plasma concentration time curve (AUC-directly proportional to the total amount of invariant drug leading to systemic circulation), the maximum (peak) plasma drug concentration, the maximum plasma The time at which the drug concentration occurs (peak time), additional factors used to accurately measure bioavailability (the compound's terminal half-life, systemic clearance, steady-state volume of distribution, And F%). These parameters are then analyzed by non-compartmental or compartmental methods to give a calculated percentage of bioavailability for an example of this type of method (for example of this type of method, Egorin et al. (See the article 2002, and references cited therein).

  The above-described compounds of the invention, or formulations thereof, can be administered by any conventional method. For example, but not limited to, these may be parenteral (such as intravenous administration), oral, topical (oral, sublingual, or transdermal), via a medical device (e.g., a stent), by inhalation, or injection (subcutaneous or Can be administered via intramuscular). The treatment consists of a single dose or multiple doses over time.

  While it is possible for a compound of the present invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. Accordingly, provided are pharmaceutical compositions comprising a compound of the present invention together with one or more pharmaceutically acceptable diluents or carriers. The diluent or carrier must be “acceptable” in the sense of being compatible with the compound of the present invention and not harmful to the recipient thereof. Examples of suitable carriers are described in further detail below.

The compounds of the present invention can be administered alone or in combination with other therapeutic agents. Co-administration of two (or more) drugs allows for a very small dose of each to be used. Thereby, side effects are reduced. Also provided is a pharmaceutical composition comprising a compound of the present invention and one or more pharmaceutically acceptable diluents or carriers together with a further therapeutic agent.
In a further aspect, the present invention provides for the use of a compound of the present invention in combination with a therapy using a second agent for the treatment of cancer or B-cell malignancy.

  In one embodiment, the compounds of the invention are co-administered with another therapeutic agent for the treatment of cancer or B-cell malignancies. Preferred drugs include but are not limited to methotrexate, leucovorin, adriamycin, prenisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate , Anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g. Herceptin (TM)), capecitabine, raloxifene hydrochloride, EGFR inhibitor (e.g. Iressa (TM), Tarceva (TM), Erbitux ™), VEGF inhibitors (eg, Avastin ™), proteasome inhibitors (eg, Velcade ™), or Glivec ™. Furthermore, the compounds of the invention can be administered in combination with other therapies such as, but not limited to, radiation therapy or surgery.

  The formulations can conveniently be given in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. Such methods include the step of bringing into association the active ingredient (the compounds of the present invention) with a carrier which constitutes one or more accessory ingredients. In general, formulations can be prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

The compounds of the present invention are usually non-toxic organic acid addition salts or organic base addition salts or inorganic acid addition salts or inorganic bases in the form of pharmaceutical preparations containing the active ingredient, usually orally or by any parenteral route. Administered in a pharmaceutically acceptable dosage form in the form of an addition salt. Depending on the disorder and patient to be treated and the route of administration, the composition can be administered at various dosages.
For example, the compounds of the present invention may be in the form of tablets, capsules, ovules, elixirs, solutions, or suspensions that may contain a flavoring or coloring agent for immediate, delayed or sustained release applications. It can be administered orally, buccally, or sublingually.

  The tablets include excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dicalcium phosphate, and glycine, starch (preferably corn, potato, or tapioca starch), sodium starch glycolate, croscarme Disintegrants such as sodium loose and certain complex silicates and granulating binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin, and acacia can be included. In addition, lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc can be included.

  Similar types of solid compositions can also be used as fillers in gelatin capsules. In this regard, preferred excipients include lactose, starch, cellulose, lactose, or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the compounds of the present invention may be combined with emulsifiers and / or suspending agents, and water, ethanol, propylene glycol, and glycerin, and combinations thereof, as well as various sweetening or flavoring, coloring Can be combined with agents or dyes.

  A tablet may be compressed or molded with one or more accessory ingredients. Compressed tablets can contain the active ingredients in a free-flowing form such as powders or granules, optionally binders (e.g. povidone, gelatin, hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives in a suitable machine. It can be prepared by mixing with an agent, a disintegrant (eg, sodium starch glycolate, crosslinked povidone, crosslinked sodium carboxymethylcellulose), a surfactant, or a dispersing agent and compressing. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets can be optionally coated or engraved, and the release or control of the active ingredients herein can be controlled using, for example, varying proportions of hydroxypropyl methylcellulose to provide the desired release characteristics. It can be formulated to provide release.

  Formulations of the present invention suitable for oral administration include as individual units such as capsules, cachets, or tablets (each containing a predetermined amount of active ingredient): as a powder or granules; solutions in aqueous or non-aqueous liquids Or as a suspension; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be provided as a bolus, electuary or paste.

Formulations suitable for topical administration in the mouth include lozenges containing the active ingredient in flavor bases, usually sucrose and acacia, or tragacanth; in inert bases such as gelatin and glycerin, or sucrose, and acacia There are pastilles containing the active ingredient; and mouthwashes containing the active ingredient in a suitable liquid carrier.
In particular, in addition to the components described above, the formulations of the present invention can include other drugs commonly used in the art, related to the type of formulation. For example, those suitable for oral administration can include perfumes.

  Pharmaceutical compositions adapted for topical administration are formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils, transdermal devices, sprays, etc. can do. These compositions can be prepared via conventional methods involving the active agent. They can therefore also contain conventional carriers and additives, such as preservatives, solvents to assist in drug penetration, emollients in creams or ointments, and ethanol or oleyl alcohol for lotions. Such carriers can be provided as about 1 to about 98% of the composition. Usually they will form about 80% or less of the composition. By way of example only, a cream or ointment comprises a sufficient amount of a hydrophilic substance containing about 5-10% by weight of the compound and water in an amount sufficient to produce a cream or ointment having the desired consistency. , By mixing.

Pharmaceutical compositions adapted for transdermal administration can be provided as individual patches intended to maintain intimate contact with the recipient's epidermis for an extended period of time. For example, the active substance is carried from the patch by iontophoresis.
For application to external tissues such as mouth and skin, preferably the composition is applied as a topical ointment or cream. When formulated in an ointment, the active substance can be used with either a paraffinic or water-miscible ointment base.

Alternatively, the active substance can be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
For parenteral administration, liquid unit dosage forms may be prepared utilizing the active ingredient and a sterile vehicle such as, but not limited to, water, alcohols, polyols, glycerin, and vegetable oils (preferably water). The active ingredient can be suspended or dissolved in the vehicle depending on the vehicle and concentration used. In preparing solutions, the active ingredient can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing.

  Advantageously, substances such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. To improve its stability, the composition can be frozen after filling into the vial and the water removed under reduced pressure. The lyophilized powder can then be sealed in a vial and an associated vial of water for injection can be provided and the liquid reconstituted prior to use.

  Parenteral suspensions can be prepared as solutions in substantially the same manner, except that the active ingredient is suspended in the vehicle instead of being dissolved and cannot be sterilized by filtration. The active ingredient can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the active ingredient.

  The compounds of the present invention can also be administered using medical devices known in the art. For example, in one embodiment, a needleless hypodermic injection device such as the device disclosed in US 5,399,163; US 5,383,851; US 5,312,335; US 5,064,413; US 4,941,880; US 4,790,824; or US 4,596,556 is used. The pharmaceutical composition of the present invention can be administered. Examples of well-known implants and modules useful in the present invention include an implantable infusion pump for dispensing drugs at a controlled rate, US 4,487,603; treatment for administering drugs through the skin US 4,486,194 disclosing device for use; US 4,447,233 disclosing a drug infusion pump for delivering a drug at a precise infusion rate; variable flow implantable infusion for continuous drug delivery US 4,447,224 disclosing an apparatus); US 4,439,196 disclosing an osmotic drug delivery system having a plurality of chamber compartments; and US 4,475,196 disclosing an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

The volume to be administered of the compounds of the invention will vary according to the particular compound, the disease involved, the subject, and the type and severity of the subject's disease and physical condition, and the route of administration chosen. Appropriate doses can be readily determined by one skilled in the art.
The composition comprises from 0.1% by weight, preferably from 5 to 60% by weight, more preferably from 10 to 30% by weight of the compound of the invention, depending on the method of administration.

  Appropriate amounts and intervals of individual administrations of the compounds of the invention will be determined by the type and extent of the condition to be treated, the mode of administration, the route of administration, the site of administration, and the age and condition of the particular subject to be treated. It will be appreciated by those skilled in the art that the physician will ultimately determine the appropriate dose to be used. This administration can be repeated an appropriate number of times. If side effects occur, the dose and / or frequency of administration can be altered or reduced according to standard clinical practice.

(General method)
(Culture fermentation)
Species Antinocinnema plethiosum subsp Prethiosum ATCC 31280 (Actinosynnema pretiosum subsp. These conditions are described in US Pat. No. 4,315,989 and US Pat. No. 4,187,292. Both strains are grown on ISP2 agar for 2-3 days at 28 ° C. (Shirling, EB and Gottlieb, D. (1966) International Journal of Systematic Bacteriology 16: 313-340) and used as inoculum seeding medium (20 parts by volume of glucose, 30 parts by volume of soluble starch, 10 parts by volume of corn steep liquor, 10 parts by volume of soy flour, 5 parts by volume of peptone, 3 parts by volume of sodium chloride, and 5 parts by volume of calcium carbonate / 1000 parts by volume of water , pH 7.0, US 4,315,989 and US 4,187,292). The inoculum seeding medium is incubated at 28 ° C. with shaking for 48 hours. For the production of macbecin and 18,21-dihydromacbecin, fermentation medium (5% glycerol, 2% corn steep liquor, 2% yeast extract, 2% KH ₂ PO ₄ , 0.5% MgCl ₂ , and 0.1% CaCO ₃ , pH 6.5, US Pat. No. 4,315,989 and US Pat. No. 4,187,292) are first incubated at 28 ° C. for 24 hours, followed by incubation time at 26 ° C. for 4-6 days. The culture is then collected for extraction.

(Extraction of culture broth for LCMS analysis)
Add culture broth (1 mL) and ethyl acetate (1 mL), mix for 15-30 minutes, then centrifuge for 10 minutes. Collect 0.5 mL of organic phase, evaporate, dry, then redissolve in 0.25 mL methanol.

(LCMS analytical procedure for fermentation broth analysis and in vivo conversion studies)
LCMS is an integrated Agilent HP1100 HPLC system combined with a Bruker Daltonics Esquire 3000+ electrospray mass spectrometer operating in positive ion mode. Chromatography was performed on a Phenomenex Hyperclone column (C ₁₈ BDS, 3u, 150 x 4.6 mm) eluting at a flow rate of 1 mL / min over 11 minutes using acetonitrile / water (40/60) to acetonitrile / water (80 / This was achieved using a linear gradient of 20).

(Synthesis)
All reactions are carried out in anhydrous conditions in oven-dried glassware cooled under vacuum using a dry solvent unless otherwise stated. The reaction is monitored by LC-UV-MS on an Agilent 1100 HPLC coupled to a Bruker Daltonics Esquire 3000 electrospray mass spectrometer, switching between positive and negative ion modes for alternating scans. Chromatography was accomplished on a Phenomenex Hyperclone column, BDS C18 3u (150 x 4.6 mm) using a linear gradient of acetonitrile: water (40: 60-100) at 1 mL / min over 11 minutes.

(Water-soluble assay)
(Dynamic measurement)
A DMSO stock solution (10 mM) of the compound was prepared. Each aliquot (0.01 mL) was made up to a total of 0.5 mL using PBS solution or DMSO. The resulting 0.2 mM solution was shaken on an IKA® vibrax VXR shaker at room temperature.

After shaking, the resulting solution or suspension was transferred to a 2 mL Eppendorf tube and centrifuged at 13200 rpm for 30 minutes. An aliquot of the supernatant was then analyzed by an Agilent 1100 HPLC coupled to a Bruker Daltonics Esquire 3000 electrospray mass spectrometer (see specific examples herein for details). Chromatography was accomplished on a Phenomenex Hyperclone column, BDS C18 3u (150 x 4.6 mm) with a linear gradient of acetonitrile / water (40 / 60-100) at 1 mL / min over 11 minutes. UV absorption was monitored at λ = 258 and 280 nm.
All analyzes were performed in triplicate, and the solubility of individual compounds was calculated by comparing their solubility in PBS using an assumed solubility of 100% in DMSO 0.2 mM.

(Thermodynamic measurement)
Mix the appropriate amount of compound in 2, 5, 10, and / or 20 mM solution with the appropriate amount of 5% glucose and DMSO in a brown glass vial and shake at room temperature on an IKA® vibrax VXR shaker. did. After 6 hours, the resulting suspension / solution was centrifuged at 13200 rpm for 20 minutes.

An aliquot of the supernatant was then analyzed by an Agilent 1100 HPLC coupled to a Bruker Daltonics Esquire 3000 electrospray mass spectrometer (see specific examples herein for details). Chromatography was accomplished on a Phenomenex Hyperclone column, BDS C18 3u (150 x 4.6 mm) with a linear gradient of acetonitrile / water (40 / 60-100) at 1 mL / min over 11 minutes. UV absorption was monitored at λ = 258 and 280 nm.
All analyzes were performed in triplicate, and the solubility of individual compounds was calculated by comparing their solubility in 5% glucose using an assumed solubility of 100% in DMSO.

(Cleavage assay for 18,21-dihydromacbecin prodrug)
Assays to measure the rate of cleavage of the prodrug to these parent compounds were performed as described herein. It was done in plasma, blood or buffer. Use mixed mouse, human, or mixed rat plasma containing EDTA or heparin as an anticoagulant, or total defibrinated rabbit blood (mouse and rat plasma, and rabbit blood are from Harlan-Sera Labs. (Human plasma was obtained from Biopredic International.)

  When using plasma, the following protocol was used. Plasma was thawed at 37 ° C. in a water bath. Plasma was dispensed into individual 20 mL tubes for each test compound so that 0.5 mL of plasma was analyzed for each time point. A tube containing plasma was also included as a control. These tubes were incubated for 15 minutes at 37 ° C.

Each test compound was reconstituted in DMSO or other suitable solvent. Plasma was removed from the water bath and the compound was added to the plasma to give a final concentration of 0.001 to 0.010 mg / mL and to keep the final concentration of solvent below 5%. For control, an equal volume of DMSO or other solvent was added. Immediately after compound addition, a 0.4 mL sample was removed from the tube and transferred to a 2 mL microtube. Plasma was quickly returned to the 37 ° C water bath. A 0.4 mL sample was removed at regular time points (eg, 30 minutes then every hour) and immediately frozen to -80 ° C.
In each experiment, the stability of the test compound in phosphate buffered saline (PBS) at a specific pH was also analyzed using the same methodology.

  After sampling at the final time point, the sample was extracted as follows. For each time point sample, an Oasis MCX (3cc / 60 mg) or Oasis HLB (333/60 mg) cartridge was prepared and equilibrated with 2 mL methanol followed by 2 mL water. 1.5 mL water and 0.002 mg / mL internal standard (IS) were added to each 0.4 mL plasma sample. This was loaded into the cartridge. The cartridge was washed with 2 mL water and the analyte was eluted with 2 ml MeOH. This extract was analyzed without further treatment by LC / MS using the following conditions.

  When using whole blood, such as defibrinated whole rabbit blood, the following protocol was used: Plasma was thawed at 37 ° C. in a water bath. Plasma was dispensed into individual 20 mL tubes for each test compound so that 0.5 mL of plasma was analyzed for each time point. A tube containing plasma was also included as a control. These tubes were incubated for 15 minutes at 37 ° C.

  Each test compound was reconstituted in DMSO or other suitable solvent. Plasma was removed from the water bath and the compound was added to the plasma to give a final concentration of 0.001 to 0.010 mg / mL and to keep the final concentration of solvent below 5%. For control, an equal volume of DMSO or other solvent was added. Immediately after compound addition, a 0.4 mL sample was removed from the tube and transferred to a 2 mL microtube. Plasma was quickly returned to the 37 ° C water bath. A 0.4 mL sample was removed at regular time points (eg, 30 minutes, then every hour) and immediately frozen to -80 ° C. To each 0.4 mL sample in the 2 mL microtube, 1.5 mL of 0.1 M potassium dihydrogen phosphate was added and inverted to mix. A stable internal standard (IS) in acetonitrile was added at 0.002 mg / mL. This was stirred for 5 minutes on an IKA shaker at 1500 rpm. Centrifugation at 5000 rpm for 5 minutes yielded a high density pellet consisting of red blood cells and precipitated protein, and a pink / red supernatant. An Oasis HLB (333/60 mg) cartridge was equilibrated with 2 mL methanol followed by 2 mL water. 1.5 mL of the supernatant was transferred to the cartridge and allowed to flow under slight positive pressure if necessary. Each cartridge was dried by flushing the cartridge with a large amount of air from a syringe and the analyte was eluted with 1 mL acetonitrile. 0.01 mL of 10% formic acid was added to the eluate and then transferred to an HPLC vial for analysis.

The different LCMS analytical procedures used to measure the cleavage rate are as follows:
(Method A)
Injection volume: 0.03 mL. The HPLC was performed on a Phenomenex Hyperclone 3 micron BDS C18 column, 150 mm x 4.60 mm with the following mobile phases:
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile Flow rate: 1 mL / min.

  HPLC conditions are: 30% B for 2 minutes, followed by a linear gradient that becomes 100% B in 14 minutes, and isocratic time at 100% B for 4 minutes. Analytes were detected by mass spectrometer using UV absorption at 255 nm and a Bruker Daltonics Esquire 3000+ mass spectrometer coupled to the HPLC. Analytes were quantified based on an extracted ion exchange chromatograph. To ensure reliable quantification, the linear response of the mass spectrometer was checked over the concentration range of interest.

(Method B)
Injection volume: 0.030 mL. HPLC was performed on a Waters Symmetry C8 3.5 micron column, 50 mm x 2.1 mm with the following mobile phases flowing:
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile Flow rate: 1 mL / min.

  HPLC conditions are: 10% B for 1 minute, followed by a linear gradient that becomes 100% B in 7 minutes, and isocratic time at 100% B for 2 minutes. Analytes were detected by mass spectrometer using UV absorption at 255 nm and a Bruker Daltonics Esquire 3000+ mass spectrometer coupled to the HPLC. Analytes were quantified based on an extracted ion exchange chromatograph. To ensure reliable quantification, the linear response of the mass spectrometer was checked over the concentration range of interest.

(Method C)
Injection volume: 0.02 mL. The HPLC was performed on a Phenomenex Hyperclone 3 micron BDS C18 column, 150 mm x 4.60 mm with the following mobile phases:
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile Flow rate: 1 mL / min.

  HPLC conditions are: 10% B for 1 minute, followed by a linear gradient that becomes 100% B in 7 minutes, and isocratic time at 100% B for 2 minutes. Analytes were detected by mass spectrometer using UV absorption at 255 nm and a Bruker Daltonics Esquire 3000+ mass spectrometer coupled to the HPLC. Analytes were quantified based on an extracted ion exchange chromatograph. To ensure reliable quantification, the linear response of the mass spectrometer was checked over the concentration range of interest.

(In vitro bioassay for anti-cancer activity)
In vitro evaluation of compounds for anticancer activity in a compartment of 12 human tumor cell lines in a monolayer proliferation assay was performed at Oncotest Testing Facility, Institute for Experimental Oncology, Oncotest GmbH, Freiburg. The characteristics of the 12 selected cell lines are summarized in Table 3.

Oncotest cell lines were established from human tumor xenografts as described in Roth et al. (1999). The origin of donor xenografts is described in Fiebig et al. (1999). Other cell lines were obtained from NCI (H460, SF-268, OVCAR-3, DU145, MDA-MB-231, MDA-MB-468) or purchased from DSMZ, Braunschweig, Germany (LNCAP).
All cell lines, unless otherwise specified, humidified atmosphere (95% air, 5% CO ₂₎ under, RPMI 1640 fold locations, 10% fetal bovine serum, and 0.1 mg / mL gentamicin (PAA, Colbe, Germany) It was grown at 37 ° C. in a “ready mix” medium containing

(Monolayer assay-brief protocol description)
Modified propidium iodide was used to evaluate the effect of test compounds on the growth of 12 human tumor cell lines (Dengler et al., (1995)).
Briefly, cells were collected from log phase cultures by trypsin treatment, counted, and cultured at a cell density depending on the cell line (5-10.000 viable cells / well) in 96 well flat bottom microtiter plates. After 24 hours of recovery when the cells resumed exponential growth, 0.010 mL of media (6 control wells per plate) or macbecin-containing media was added to the wells. Each concentration was cultured three times. Compounds were added at two concentrations (0.001 mM and 0.01 mM). After 4 days of continuous exposure, the cell culture medium with or without the test compound was replaced with 0.2 mL propidium iodide (PI) solution (7 mg / L). To determine the percentage of viable cells, the cells were permeabilized by freezing the plates. After plate thawing, fluorescence was measured using a Cytofluor 4000 microplate reader (excitation 530 nm, emission 620 nm). This gives a direct relationship to the total number of viable cells.
Growth inhibition was expressed as treatment / control × 100 (% T / C).

(In vivo evaluation of anti-tumor effects-brief description of protocol)
PRXF DU-145 is a prostate cancer cell line that was first isolated from a metastatic central nervous system lesion of a 69 year old prostate cancer elderly person. PRXF DU-145 cell suspension was injected subcutaneously into nude mice and the resulting xenografts were passaged into nude mice until a stable growth pattern was established.

  Tumor fragments were obtained from serial passage xenografts of nude mice. After removal of donor mouse-derived tumors, they were cut into fragments (line 1-2 mm) and RPMI 1640 medium (RPMI 1640 medium containing 25 mM HEPES buffer with L-glutamine, Gibco, catalog # 52400-025) Incubated until subcutaneous implantation. Recipient mice were anesthetized by inhalation of isoflurane. A small cut was made in the skin on the back for bilateral implantation. Tumor fragments were transplanted with tweezers.

  Randomly, tumor-bearing animals were stratified into treatment and control groups according to tumor volume using “Lindner's Randomization Tables”. Only animals with appropriately sized tumors (average tumor diameter: 6-8 mm, minimum acceptable tumor diameter: 5 mm) were considered for randomization. When the maximum number of mice were found to be randomized, mice were randomized. The day of randomization was represented as day 0. Day 0 is also the first day of administration. Next, tumor growth was compared between mice given different dosing regimens. Mice were monitored daily with an observation period of 42 days.

Tumor volume was determined by two-dimensional measurements with a caliper on the day of randomization (day 0) followed by twice a week. Tumor volume was calculated according to the formula: (ax b2) x 0.5. Where a represents the largest portion and b represents its vertical tumor diameter.
Antitumor activity was evaluated as the maximum tumor volume inhibition relative to the vehicle control group.

グループ腫瘍容積は、グループにおいて全腫瘍のメジアンRTVとして表した(グループメジアンRTV)。グループメジアンRTV値は、増殖曲線を描くため、及び処置評価のために使用した。 The relative volume of individual tumors (RTV) was calculated by dividing the individual tumor volume on day X (Tx) by the individual tumor volume on day 0 and multiplying by 100%.

Group tumor volume was expressed as median RTV of all tumors in the group (group median RTV). Group median RTV values were used to draw growth curves and for treatment evaluation.

For each group, tumor inhibition (T / C%) on a particular day was calculated by multiplying the ratio of the median RTV value between the individual test group and the vehicle control group by 100%.

The maximum (or optimal) T / C% value recorded for a particular test compound during the experimental period represents the maximum antitumor activity for each treatment.
A U test by Mann-Whitney Wilcoxon was performed to assess the statistical significance of tumor inhibition. The assay compares the ranking of individual tumors on the one hand according to the relative capacity of the vehicle control group and on the other hand according to the relative capacity of the test group of interest. By convention, a p value <0.05 indicates the significance of tumor inhibition.

(Intermediate 1: Production of macbecin using antinocinnema mirum DSM 43827 (Actinosynnema mirum DSM 43827) and antinocinnema prethiosum subsp prethiosum ATCC 31280 (Actinosynnema pretiosum subsp pretiosum ATCC 31280) in falcon tubes
Falcon tubes containing 10 mL of seeding medium were inoculated using agar plugs cut from high density A. Milmlawn on ISP2 agar and incubated as described in the general method. Seed culture (0.5 mL) was used for inoculation of 10 mL of production medium, followed by incubation and extraction as described in the general method. Analysis by LCMS showed the presence of macbecin eluting at 9.5 minutes ([M + Na] ⁺ , m / z = 581.3).

(Intermediate 1 (another preparation method): Macbecin production using Antinocinnema mirum DSM 43827 in 15 liter fermenter)
Five 2 L conical shake flasks were prepared, each with 300 mL seeding medium, and agar plugs cut from A. Milm's densely grown lawn were inoculated at 15 per flask. The culture was shaken at 28 ° C. for 48 hours. These seeding cultures were used to inoculate a fermentation vessel containing 15 L of production medium (10% inoculation). Fermentation was performed using an impeller tip speed of 1.18 m / s to 2.75 m / s, 1 vvm aeration; these were heated at 28 ° C. for 24 hours and subsequently lowered to 24 ° C. At the start of fermentation, the pH was adjusted to pH 6.5-7 using 1 MH ₂ SO ₄ and 1 M NaOH. The baffle was tilted to 45 ° and the impeller tip speed was adjusted according to the oxygen demand (maximum dissolved oxygen was maintained at 30%). Antifoam SAG471 was added at 0.2% v / v before and after autoclaving as needed at the start of fermentation. The fermentor was collected after 230 hours. Analysis by LCMS indicated the presence of macbecin eluting after 9.5 minutes ([M + Na] ⁺ , m / z = 581.3).

  The fermentation broth was centrifuged at 3500 rpm for 30 minutes to separate the cells from the supernatant. The supernatant was then distributed 3 times with an equal volume of ethyl acetate. The cell pellet was extracted twice with an equal volume of acetone. The organic fractions were combined and the solvent was removed under reduced pressure. The resulting aqueous slurry was extracted three times with an equal amount of ethyl acetate, and the combined organic fractions were concentrated to give a crude extract.

To the crude extract was added flash silica gel previously dissolved in acetone, followed by removal of the solvent under reduced pressure. Permeated silica was added to an open column of flash silica pre-prepared with hexane. This is a step gradient of hexane: ethyl acetate (100: 0, 80:20, 75:25, 70:30, 50:50, 0: 100) and finally ethyl acetate: methanol (50:50, 0: 100 ). The volume of each step fraction was about 1 liter of solvent mixture. Fractions containing macbecin were combined, evaporated and dried. Further purification was performed on a reverse phase HPLC (Phenomenex Luna column (C18, 10 micron, 250 x 5u, 250 x 21.20 mm)) eluent A (acetonitrile: water, 20:80) to eluent B (acetonitrile). And eluting over 30 minutes at a flow rate of 21 mL / min. Fractions containing macbecin were combined and concentrated to give a yellow powder (45 mg). The structure of Macbecin (CDCl ₃ ) was confirmed by multidimensional NMR spectroscopy using a Bruker Advance 500 MHz freezing instrument (see Table 4). This is consistent with published data (Muroi et al. 1981).

(Example 1)
(18-O- (N, N'-Dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, Synthesis of 1 (Route 1))
(Conversion of macbecin to 18,21-dihydromacbecin)
Macbecin (107.8 mg, 0.193 mmol) was dissolved in ethyl acetate (25 mL) and treated with 96 mM sodium hydrosulfite solution (3 × 5 mL). In each case, the phases were mixed vigorously in a separatory funnel and the aqueous phase was removed. The organic phase goes from dark yellow to substantially colorless. The organic phase is then washed with water (3 x 10 mL), dried over anhydrous sodium sulfate, filtered, and the solvent removed under reduced pressure to give 18,21-dihydromacbecin as an off-white glassy solid. (105.0 mg, 0.187 mmol, 97% isolated yield). 18,21-Dihydromacbecin was used without further purification.
LCMS: Macbecin, RT = 8.2 min ([MH] ^- , m / z = 557.5, [M + Na] ⁺ , m / z = 581.2) UV λ _max = 256 (sh) nm; 18,21-dihydromacbecin , RT = 3.5 min ([MH] ⁻ , m / z = 559.5, [M + Na] ⁺ , m / z = 583.3) UV λ _max = 302 nm.

(Preparation of N-tert-butoxycarbonyl-N, N'-dimethylethylenediamine)
N, N′-dimethylethylenediamine (1.0 g, 11.3 mmol) was dissolved in anhydrous dichloromethane (10 mL) and treated with triethylamine (1.6 mL, 11.3 mmol). The mixture was cooled to 0 ° C. for the addition of di-tert-butyl dicarboxylate (2.5 g, 11.3 mmol). The reaction was stirred for 30 minutes at 0 ° C., followed by 2 hours at room temperature. The reaction mixture was then washed with water (10 mL) and the aqueous phase was extracted with a further portion of dichloromethane (2 × 10 mL). The combined organic phases were dried over Na ₂ SO ₄ and the solvent was removed under reduced pressure. Purification by column chromatography (40: 8: 1, dichloromethane: methanol: aqueous ammonia) gave the desired N-tert-butoxycarbonyl-N, N′-dimethylethylenediamine as a colorless oil (508 mg, 24% ).

(18-O-[(N-tert-butoxycarbonyl) -N, N'-dimethylethylenediamine-N'-carbamoyl] -18,21-dihydromacbecin to obtain N- (tert-butoxycarbonyl)- (O-acylation of 18,21-dihydromacbecin with N, N'-dimethylethylenediamine)
18,21-Dihydromacbecin (2.00 mg, 3.6 × 10 ⁻³ mmol) was dissolved in anhydrous degassed dichloromethane (1 mL). 4-Nitrophenyl chloroformate (0.86 mg, 4.3 × 10 ⁻³ mmol) was added, followed by 2,6-lutidine (2.48 × 10 ⁻³ mL, 21.4 × 10 ⁻³ mmol). The reaction mixture was heated to reflux for 4 hours to form an intermediate carbonate. N-tert-butoxycarbonyl-N, N′-dimethylethylenediamine (2.70 mg, 14.2 × 10 ⁻³ mmol) was added and the reaction was heated and refluxed for a further 2 hours. The reaction mixture was washed with water (2 mL) and the crude material was analyzed. The data corresponded to the data for the desired 18-O-[(N-tert-butoxycarbonyl) -N, N′-dimethylethylenediamine-N′-carbamoyl] -18,21-dihydromacbecin.
LCMS: 18-O-[(N-tert-butoxycarbonyl) -N, N'-dimethylethylenediamine-N'-carbamoyl] -18,21-dihydromacbecin, RT = 6.8 min ([MH] ^- , m / z = 773.4), UV λ _max = 266 nm.

18-O-[(N-tert-butoxycarbonyl) -N, to obtain (18-O- (N, N'-dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, N'-Dimethylethylenediamine-N'-carbamoyl] -18,21-dihydromacbecin deprotection)
18-O-[(N-tert-butoxycarbonyl) -N, N'-dimethylethylenediamine-N'-carbamoyl] -18,21-dihydromacbecin (20 mg, 25.8 μmol) was dissolved in diethyl ether Treated with excess 2 M HCl until the starting material was consumed. The residue was purified by tituration with diethyl ether to obtain 18-O- (N, N'-dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride (11 mg, 60% ) Was obtained as a pale yellow solid.
Direct injection MS: 18-O- (N, N'-dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin, [MH] ⁺ , m / z = 673.5, [M + Na] ⁺ , m / z = 697.4, [M + NH] ⁺ , m / z = 675.4.

(Example 2)
(18-O- (N, N'-Dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, synthesis of 1 (route 2))
(Preparation of 18-O- (4-nitrophenyl carbonate) -18,21-dihydromacbecin)
Macbecin II (0.30 g, 0.54 mmol) was dissolved in anhydrous dichloromethane (72 ml). To this solution was added 4-nitrophenyl chloroformate as a solid (0.183 g, 0.91 mmol) followed by 2,6-lutidine (0.217 ml, 1.87 mmol). The reaction mixture was heated to reflux at 50 ° C. (oil bath) for 5 hours under argon. The reaction was cooled to ambient temperature and subsequently washed with an equal volume of 1N HCl and water, dried over Na ₂ SO ₄ and filtered, and the solvent was removed under reduced pressure. The resulting material was purified on silica gel eluting with a step gradient of acetone in hexane (5-40% acetone, increasing with 5% increase) to give the title compound. Isolated yield: 0.310 g (79%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound.
LCMS: RT = 7.2 min ([MH] ^- , m / z = 724.0).

(Preparation of N-trityl-N, N'-dimethylethylenediamine)
N, N′-dimethylethylenediamine (5.0 g, 56.7 mmol) was dissolved in dichloromethane (20 ml) under argon followed by treatment with trimethylamine (5.74 g, 56.7 mmol). The stirred mixture was cooled to 0 ° C., and a solution of trityl chloride (15.82 g, 56.7 mmol) in dichloromethane (20 mL) was slowly added dropwise. After the addition of this solution was complete, the reaction mixture was stirred at 0 ° C. for another 30 minutes. At that time, the cooling bath was removed and the reaction was allowed to warm to room temperature. The mixture was stirred overnight at room temperature under argon. The resulting solution was partitioned between dichloromethane and water and a white precipitate of the product HCl salt was observed. This mixture was treated with 10% aqueous K ₂ CO ₃ (100 mL) resulting in dissolution of a white solid and partitioning into two phases. The two phases were separated and the aqueous phase was extracted with dichloromethane (2 × 200 mL). The combined organic extracts were dried over MgSO ₄ and the solvent was removed under reduced pressure. The residue was purified on silica gel eluting with dichloromethane: methanol: aqueous ammonia (80: 5: 0.5). The purified fraction was collected and the solvent was removed under reduced pressure. The remaining water / ammonia was removed by addition of isopropyl alcohol followed by removal under reduced pressure (3 × 300 ml) and then under high vacuum to give the title compound as a pale yellow solid. Isolated yield, 6.30 g (34%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound.

(Preparation of 18-O- (N-trityl-N, N'-dimethylethylenediamine-N'-carbamoyl) -18-21-dihydromacbecin)
18-O- (4-nitrophenyl carbonate) -18,21-dihydromacbecin (0.89 g, 1.23 mmol) was dissolved in dichloromethane (40 mL). Then N-trityl-N, N′-dimethylethylenediamine (1.21 g, 3.68 mmol) in dichloromethane (40 mL) was added and the solution was heated to reflux for 2 hours followed by stirring overnight at room temperature. TLC indicated the presence of starting material and the mixture was refluxed for an additional 2 hours. It was then cooled to room temperature, washed with water, dried over Na ₂ SO ₄ and the solvent removed under reduced pressure. The resulting material was purified by chromatography on silica gel eluting with hexane: acetone (5: 3) to give a pale green solid. Isolated yield: 0.92 g (82%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound.
LCMS: RT = 11.1 min ([MH] ^- , m / z = 915.6 (weak); [MH-trityl] ^- , m / z = 673.4; [M + H-trityl] ⁺ , m / z = 675.5).

(Preparation of 18-O- (N, N'-dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, 1)
18-O- (N-trityl-N, N'-dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin (0.10 g, 0.11 mmol) dissolved in anhydrous dichloromethane (40 mL) under argon And cooled to −5 ° C. using an ice / salt bath. 2 M HCl in ether (0.104 mL, 0.21 mmol) was dissolved in anhydrous dichloromethane (0.2 mL), followed by dropwise addition of the cooled substrate solution. The reaction was stirred for 5 minutes at -5 ° C followed by 90 minutes at room temperature. Hexane (40 mL) was added to precipitate the product salt and remaining starting material. The precipitate was triturated and subsequently washed twice with cold ether to remove all starting material and other impurities. Isolated yield: 0.060 g (77%).
LCMS: RT = 3.1 min ([MH] ⁻ , m / z = 673.5; [M + H] ⁺ , m / z = 675.4).

Example 3
(Synthesis of 18-O- (N-methylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, 2)
(Preparation of N-trityl-N-methylethylenediamine)
N-methylethylenediamine (5.96 g, 80.41 mmol) was dissolved in dichloromethane (100 mL) under argon. The stirred mixture was cooled to 0 ° C., and a solution of trityl chloride (6.47 g, 23.21 mmol) in dichloromethane (40 ml) was slowly added dropwise. After the addition of this solution was complete, the reaction mixture was stirred at 0 ° C. for another 30 minutes. At this point, the cooling bath was removed and the reaction was allowed to warm to room temperature. The mixture was stirred overnight at room temperature under argon. The solvent was removed from the resulting solution, and ethyl acetate (200 mL) and saturated aqueous NaHCO ₃ (200 mL) were added. The resulting mixture was shaken and separated, and the aqueous phase was further extracted with an equal volume of ethyl acetate. The combined organic phases were dried over Na ₂ SO ₄ , filtered and the solvent removed under reduced pressure. The residue was purified on silica gel eluting with dichloromethane: methanol: aqueous ammonia (80: 5: 0.1). Two regioisomers were observed and separated individually. The purified fraction of the title compound (more polar on silica) was collected and the solvent was removed under reduced pressure. Isolated yield: 1.66 g (22%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound and the correct regioisomeric correspondence was observed.

(Preparation of 18-O- (N-trityl-N-methylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin)
18-O- (4-Nitrophenyl carbonate) -18,21-dihydromacbecin (0.100 g, 0.14 mmol) was dissolved in dichloromethane (20 ml). N-trityl-N-methylethylenediamine (0.131 g, 0.41 mmol) was then added and the solution was heated to reflux for 2 hours followed by stirring overnight at room temperature. The organic phase was washed with water (20 mL) followed by drying over anhydrous Na ₂ SO ₄ , filtering and removing the solvent under reduced pressure. The material was purified by chromatography on silica gel eluting with hexane: acetone (7: 3). Isolated yield: 0.093 g (74%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound.
LCMS: RT = 11.2 min ([MH] ^- , m / z = 901.4).

(Preparation of 18-O- (N-methylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, 2)
18-O- (N-trityl-N-methylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin (0.093 g, 0.10 mmol) was dissolved in anhydrous dichloromethane (19.5 mL) under argon and iced. / Cooled to -5 ° C using a salt bath. Next, 2M HCl in ether (0.098 mL, 0.196 mmol) was added dropwise to the cooled substrate solution. The reaction was stirred for 5 minutes at -5 ° C followed by 60 minutes at room temperature. Hexane (20 mL) was added to precipitate the product salt and remaining starting material. The precipitate was triturated and subsequently washed twice with cold ether to remove all starting material and other impurities. The resulting solid was recrystallized from dichloromethane: ether. Isolated yield: 0.021 g (29%).
LCMS: 9.7 min ([MH] ^- , 658.9).

Example 4
(Synthesis of 18-O- (N, N'-diethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, 3)
(Preparation of N-trityl-N, N'-diethylethylenediamine)
N, N′-diethylethylenediamine (2.5 g, 21.5 mmol) was dissolved in dichloromethane (20 ml) under argon. The stirred mixture was cooled to 0 ° C. and a solution of trityl chloride (2.4 g, 8.6 mmol) in dichloromethane (20 mL) was slowly added dropwise. After the addition of this solution was complete, the reaction mixture was stirred at 0 ° C. for another 30 minutes. At this point, the cooling bath was removed and the reaction was allowed to warm to room temperature. The mixture was stirred overnight at room temperature under argon. The solvent was removed from the resulting solution, and ethyl acetate (200 mL) and saturated aqueous NaHCO ₃ solution (200 mL) were added. The resulting mixture was shaken and separated, and the aqueous phase was further extracted with an equal volume of ethyl acetate. The combined organic phases were dried over Na ₂ SO ₄ , filtered and the solvent removed under reduced pressure. The residue was purified on silica gel eluting with dichloromethane: methanol: aqueous ammonia (80: 5: 0.5). The purified fraction was collected and the solvent was removed under reduced pressure. The remaining water / ammonia was removed by addition of isopropyl alcohol followed by removal under reduced pressure (2 × 100 ml) and then under high vacuum to give the title compound as a pale yellow solid. Isolated yield: 2.60 g (84%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound.

(Preparation of 18-O- (N-trityl-N, N'-diethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin)
18-O- (4-nitrophenyl carbonate) -18,21-dihydromacbecin (0.296 g, 0.41 mmol) was dissolved in dichloromethane (15 mL). Then N-trityl-N, N′-diethylethylenediamine (0.438 g, 1.22 mmol) in dichloromethane (15 mL) was added and the solution was heated to reflux for 2 hours and stirred at room temperature overnight. The resulting solution was washed with water, dried over Na ₂ SO ₄ and the solvent was removed under reduced pressure. The resulting material was purified by chromatography on silica gel eluting with a step gradient of acetone in hexane (5-25% acetone) to give a pale yellow solid. Isolated yield: 0.23 g (59%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound.
LCMS: The material was cleaved under mobile phase conditions, lost the trityl group and liberated the secondary amine. RT = 4 min (broad) ([MH] ^- , m / z = 701.6; [M + H] ⁺ , m / z = 703.6).

(Preparation of 18-O- (N, N'-diethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, 3)
18-O- (N-trityl-N, N'-diethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin (0.206 g, 0.22 mmol) dissolved in anhydrous dichloromethane (40 mL) under argon And cooled to −5 ° C. using an ice / salt bath. 2M HCl in ether (0.207 mL, 0.41 mmol) was dissolved in anhydrous dichloromethane (1 mL) and then added dropwise to the cooled substrate solution. The reaction was stirred for 5 minutes at -5 ° C followed by 90 minutes at room temperature. Hexane (50 mL) was added to precipitate the product salt and remaining starting material. The precipitate was triturated and subsequently washed twice with cold ether to remove all starting material and other impurities. Isolated yield: 0.090 g (55%).
LCMS: RT = 3.4 min ([MH] ⁻ , m / z = 701.6; [M + H] ⁺ , m / z = 703.6).

(Example 5)
(Synthesis of 18-O- (N, N'-dimethyl-1,3-propanediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, 4)
(Preparation of N-trityl-N, N'-dimethyl-1,3-propanediamine)
N, N′-dimethyl-1,3-propanediamine (2.50 g, 24.5 mmol) was dissolved in dichloromethane (20 mL) under argon. The stirred mixture was cooled to 0 ° C. and a solution of trityl chloride (2.73 g, 9.80 mmol) in dichloromethane (20 mL) was added slowly dropwise. After the addition of this solution was complete, the reaction mixture was stirred at 0 ° C. for another 30 minutes. At this point, the cooling bath was removed and the reaction was allowed to warm to room temperature. The mixture was stirred overnight at room temperature under argon. The solvent was removed from the resulting solution, and ethyl acetate (200 mL) and saturated aqueous NaHCO ₃ solution (200 mL) were added. The resulting mixture was shaken and separated, and the aqueous phase was further extracted with an equal volume of ethyl acetate. The combined organic phases were dried over Na ₂ SO ₄ , filtered and the solvent removed under reduced pressure. The residue was purified on silica gel eluting with dichloromethane: methanol: aqueous ammonia (80: 5: 0.5). The purified fraction was collected and the solvent was removed under reduced pressure. Residual water / ammonia was removed by addition of ethanol (2 × 100 mL) followed by removal at 50 ° C. under reduced pressure, then dried under high vacuum to give the title compound. Isolated yield: 2.60 g (76%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound.

(Preparation of 18-O- (N-trityl-N, N'-dimethyl-1,3-propanediamine-N'-carbamoyl) -18,21-dihydromacbecin)
18-O- (4-nitrophenyl carbonate) -18,21-dihydromacbecin (0.161 g, 0.22 mmol) was dissolved in dichloromethane (15 mL). Next, N-trityl-N, N′-dimethyl-1,3-propanediamine (0.229 g, 0.67 mmol) in dichloromethane (15 mL) was added and the solution was heated to reflux for 3.5 hours, followed by room temperature. Stir overnight. The resulting solution was washed with water, dried over Na ₂ SO ₄ and the solvent was removed under reduced pressure. The resulting material was purified by chromatography on silica gel eluting with a step gradient of acetone in hexane (30-50% acetone) to give a white solid that covered. Isolated yield: 0.080 g (37%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound.
LCMS: RT = 8.3 min ([MH-trityl] ^- , m / z = 687.5; [M + H-trityl] ⁺ , m / z = 689.5); the material is also cleaved and released under mobile phase conditions The amine was liberated. RT = 3.2 min ([MH-trityl] ⁻ , 687.5; [M + H-trityl] ⁺ , 689.5).

(Preparation of 18-O- (N, N'-dimethyl-1,3-propanediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, 4)
18-O- (N-trityl-N, N'-dimethyl-1,3-propanediamine-N'-carbamoyl) -18,21-dihydromacbecin (0.075 g, 0.08 mmol) was dissolved in anhydrous dichloromethane under argon (8 mL) and cooled to −5 ° C. using an ice / salt bath. 2M HCl in ether (0.079 mL, 0.16 mmol) was dissolved in anhydrous dichloromethane (0.5 mL) and then added dropwise to the cooled substrate solution. The reaction was stirred for 5 minutes at -5 ° C followed by 90 minutes at room temperature. Hexane (20 mL) was added to precipitate the product salt and remaining starting material. The precipitate was triturated and subsequently washed twice with cold ether to remove all starting material and other impurities. Isolated yield: 0.038 mg (65%).
LCMS: RT = 3.3 min ([MH] ^- , m / z = 687.7; [M + H] ⁺ , 689.7).

Example 6
(Synthesis of 18-O- (N, N'-diisopropylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, 5)
(Preparation of 18-O- (N-trityl-N, N'-diisopropylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin)
18-O- (4-nitrophenyl carbonate) -18,21-dihydromacbecin (0.161 g, 0.22 mmol) was dissolved in dichloromethane (15 mL). Then N-trityl-N, N′-diisopropylethylenediamine (0.257 g, 0.67 mmol) in dichloromethane (15 mL) was added and the solution was heated to reflux for 3.5 hours followed by stirring overnight at room temperature. The resulting solution was washed with water, dried over Na ₂ SO ₄ and the solvent was removed under reduced pressure. The resulting material was purified by chromatography on silica gel eluting with a step gradient of acetone in hexane (30-50% acetone) to give a pale yellow solid. Isolated yield: 0.060 g (28%). The NMR spectrum obtained in CDCl ₃ at 400 MHz was consistent with the title compound.
LCMS: RT = 7.8 min [MH-trityl] ^- , m / z = 729.6; [M + H-trityl] ⁺ , m / z = 731.6 .; the substance is also cleaved under mobile phase conditions Liberates the primary amine. RT = 5.7 min ([MH] ^- , m / z = 729.6; [M + H] ⁺ , m / z = 731.6).

(Preparation of 18-O- (N, N'-diisopropylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride, 5)
18-O- (N-trityl-N, N'-diisopropylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin (0.055 g, 0.06 mmol) dissolved in anhydrous dichloromethane (10 mL) under argon And cooled to −5 ° C. using an ice / salt bath. 2M HCl in ether (0.055 mL, 0.11 mmol) was dissolved in anhydrous dichloromethane (1 mL) and then added dropwise to the cooled substrate solution. The reaction was stirred at −5 ° C. for 5 minutes followed by 90 minutes at room temperature. Hexane (20 mL) was added to precipitate the product salt and remaining starting material. The precipitate was triturated and subsequently washed twice with cold ether to remove all starting material and other impurities. Isolated yield: 0.032 mg (70%).
LCMS: RT = 5.5 min ([MH] ⁻ , m / z = 729.9; [M + H] ⁺ , 731.9).

(Example 7)
(Cleavage assay of compound 1 in human plasma)
Compound 1 was incubated in human plasma as described in the general method-cleavage assay above. Aliquots were taken every 15 minutes and acidified by the addition of 0.01 mL phosphoric acid to stop the chemically generated cleavage of the parent compound. Subsequently, samples were extracted as described above and immediately analyzed using analytical method B. The decay of the parent compound and the relative amounts of related compounds are shown in FIG. Here, white circles indicate an attenuation of 1, and black squares and black triangles indicate the accumulation of macbecin and 18,21-dihydromacbecin, respectively.

(Example 8)
(Comparison of cleavage rates of compounds 1-5 in phosphate buffer and whole blood)
Each compound was evaluated for cleavage to 18,21-dihydromacbecin as follows. Compounds 1-5 were incubated in whole defibrinated rabbit blood as described in the general method-cleavage assay above. Aliquots were taken at 2 minutes, 1 hour, 2.25 hours, and 3.5 hours. 1.5 mL of potassium dihydrogen phosphate was added to bring the internal standard (IS) to 0.002 mg / mL. The sample was applied to the cartridge described above and analyzed immediately using analytical method C. T1 / 2 values were calculated and are shown in Table 5.
Cleavage of compounds 1-3 was also measured in phosphate buffer at pH 7.2 and pH 7.4 as described in the general method-cleavage assay above. T _1/2 values were calculated as shown in Table 5 below.

Example 9
(Measurement of solubility of compounds 1 to 3)
Compounds 1-3 were tested for their thermodynamic solubility using the method described in General Methods. The results are shown in Table 6 below.

(Example 10)
(Biological data-in vitro evaluation of anticancer activity of macbecin)
In vitro evaluation of macbecin for anticancer activity in compartments of 12 human tumor cell lines in a monolayer proliferation assay was performed using a modified propidium iodide assay as described in General Methods.
The results are shown in Table 7 below: Each result represents the average of two repeated experiments.

Example 11
(Biological data-in vivo evaluation of 18-O- (N, N'-dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride (Compound 1))
In vivo evaluation of 18-O- (N, N′-dimethylethylenediamine-N′-carbamoyl) -18,21-dihydromacbecin hydrochloride 1 as described in General Methods, Human Prostate Cancer Cell Line Performed in nude mice with DU-145 xenografts. 18-O- (N, N'-dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride 1 is administered at a dosage level of 60 mg / kg / d in 5% glucose vehicle. -4, 7-11, 14-18, 21, 24, 25, 28, and 29, intraperitoneally administered with standard vehicle 10% DMSO, 0.05% Tween 80 Compared to the group.

  18-O- (N,) administered ip at 60 mg / kg / d on days 0-4, 7-11, 14-18, 21, 24, 25, 28, and 29 N'-dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin hydrochloride 1 significantly reduced tumor growth rate (minimum T / C value: 24.3%, recorded at 31 days) (p <0.001; U test by Mann Whitney Wilcoxon). A graph showing the median RTV value of the group over the course of this study is shown in FIG.

  Anti-tumor activity was 10% DMSO, 0.05% Tween80 in standard vehicle PBS at 10 mL / kg / d 0-4 days, 7-11 days, 14-18 days, 21 days, 24 days, 25 days, 28 days And tumor volume inhibition relative to the control group tumor received intraperitoneally on day 29. Group size was 6 mice for the vehicle control group and 6 mice in the treatment group transplanted with 2 tumor fragments. The experiment ended in 42 days.

All references cited in this application, including patents and patent applications, are incorporated herein by reference for the full extent possible.
Throughout this specification and the claims that follow, the words “comprise” and variants such as “comprises” and “comprising” require other contexts. Unless otherwise indicated, it will be understood that it does not mean the exclusion of any other integer or stage, or group of integers or stages, but the inclusion of a specified integer or stage, or the inclusion of an integer group.

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Graph showing the decay of the parent compound in the cleavage assay and the relative amount of related compounds. Graph showing group median relative tumor volume over time in PRXF DU-145 xenografts.

Claims (34)

  A derivative of a benzenoid ansamycin comprising a 1,4-dihydroxyphenyl moiety having an aminocarboxy substituent at the 6-position, wherein the 2- and 6-position carboxy substituents are linked by a variable length aliphatic chain. , One or both of the 1-hydroxy and 4-hydroxy positions of the phenyl ring are independently an aminoalkyleneaminocarbonyl group (the alkylene group can optionally be substituted with an alkyl group and have 2 to 3 carbon atoms). Characterized by having a chain length) and the derivatized group increases the water solubility and / or bioavailability of the parent molecule but can be removed in vivo. Said derivative. A compound of the following formula (IA-IC) or a pharmaceutically acceptable salt thereof:

(Where
R ₁ represents H, OH, OMe, —NHCH ₂ CH═CH ₂ , or —NHCH ₂ CH ₂ N (CH ₃ ) ₂ ;
R ₂ represents OH or keto;
R ₃ represents OH or OMe;
R ₅ is H, or

(Where
n represents 0 or 1;
R ₆ represents H, Me, Et, or iso-propyl;
R ₇ , R ₈ , and R ₉ each independently represent H, or a C1-C4 branched or straight chain alkyl group; or R ₇ and R ₈ , or R ₈ and R ₉ are 6-membered carbons Can be linked to form a ring;
R ₁₀ represents H, or a C1-C4 branched chain or straight chain alkyl group. );
However, if both R ₅ sites are not H and neither R ₅ is H, the two R ₅ sites are the same. ). R ₆ is H, Me, or an Et, compound of claim 2. The compound according to claim 2, wherein R ₁₀ represents a C1-C4 branched or straight chain alkyl group. R ₆ is H, Me, or represents Et, and R ₁₀ represents C1-C4 branched or straight chain alkyl group The compound of claim 2, wherein. 6. A compound according to any one of claims 2 to ₅ , wherein neither R5 is H. 6. A compound according to any one of claims 2 to _{5, wherein} one R5 group represents H. R ₅ groups of C21 is H, 7. Compounds according.   9. A compound according to any one of claims 2 to 8, defined by structure (IA).   9. A compound according to any one of claims 2 to 8, defined by structure (IB). R ₁ represents -NHCH ₂ CH = CH _2, wherein compound of claim 10. R ₁ represents _{-NHCH 2 CH 2 N (CH 3} ) 2, wherein compound of claim 10. 11. A compound according to claim 10, wherein R < ₁ > represents OMe.   9. A compound according to any one of claims 2 to 8, defined by structure (IC).   15. A compound according to any one of claims 2 to 14, wherein n is 0. R ₆ represents Me, any one compound according to claim 2-15. R ₆ represents Et, any one compound according to claim 2-15. R ₁₀ represents Me, any one compound according to claim 2 to 17. R ₁₀ represents Et, any one compound according to claim 2 to 17. R ₇ represents H, any one compound according to claim 2 to 19. R ₈ and R ₉ represents H, any one compound according to claim 2-20.   The compound according to claim 2, which is 18-O- (N, N'-dimethylethylenediamine-N'-carbamoyl) -18,21-dihydromacbecin, or a pharmaceutically acceptable salt thereof. The compound of claim 2 selected from:
18-O- (N-methylethylenediamine-N′-carbamoyl) -18,21-dihydromacbecin;
18-O- (N, N′-diethylethylenediamine-N′-carbamoyl) -18,21-dihydromacbecin;
18-O- (N, N′-dimethyl-1,3-propanediamine-N′-carbamoyl) -18,21-dihydromacbecin; and
18-O- (N, N′-diisopropylethylenediamine-N′-carbamoyl) -18,21-dihydromacbecin,
Or a pharmaceutically acceptable salt of any one of them.   24. A compound according to any one of claims 1 to 23 in the form of a hydrochloride salt.   25. A compound according to any one of claims 1 to 24 for use as a medicament.   25. A compound according to any one of claims 1 to 24 for the use of a medicament in the treatment of cancer or B-cell malignancy.   25. A pharmaceutical composition comprising a compound according to any one of claims 1 to 24 together with one or more pharmaceutically acceptable diluents or carriers.   25. A method for treating cancer or B-cell malignancy comprising administering to a patient an effective amount of a compound according to any one of claims 1-24.   Use of a compound according to any one of claims 1 to 24 in the manufacture of a medicament for the treatment of cancer or B-cell malignancy. A process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof:
(a) a compound of formula (IIA), (IIB), or (IIC), or a protected derivative thereof

Wherein R ₁ , R ₂ and R ₃ are as defined in any one of claims _{2 to} 23 and L is a leaving group.
Compound of formula (H)

(In the formula, n, R ₆ , R ₇ , R ₈ , R ₉ and R ₁₀ are as defined in any one of claims 2 to 23, and P represents a protecting group.)
Preparing a compound of formula (I) in which neither of the R ₅ sites is H by reaction with: or
(b) a compound of formula (IID), (IIE), or (IIF) or a protected derivative thereof

Wherein R ₁ , R ₂ and R ₃ are as defined in any one of claims _{2 to} 23 and L is a leaving group.
Compound of formula (H)

(In the formula, n, R ₆ , R ₇ , R ₈ , R ₉ and R ₁₀ are as defined in any one of claims 2 to 23, and P represents a protecting group.)
By reaction with, it R ₅ sites C21 to prepare compounds of formula (I) is H; or
(c) converting a compound of formula (I) or a salt thereof into another compound of formula (I) or another pharmaceutically acceptable salt thereof; or
(d) said method comprising deprotecting the protected compound of formula (I). A compound of (IIA), (IIB), or (IIC), or any one protected derivative thereof:

(Wherein R ₁ , R ₂ , and R ₃ are as defined in any one of claims _{2 to} 23 and L is a leaving group). A compound of (IID), (IIE), or (IIF), or any one protected derivative thereof:

(Wherein R ₁ , R ₂ , and R ₃ are as defined in any one of claims _{2 to} 23 and L is a leaving group). A compound of (IVA), (IVB), or (IVC), or any one protected derivative thereof:

(Wherein R ₁ , R ₂ , R ₃ and R ₅ are as defined in any one of claims 2 to 23 and Pa is a protecting group). A compound of (VA), (VB), or (VC), or any one protected derivative thereof:

(Wherein R ₁ , R ₂ , and R ₃ are as defined in any one of claims _{2 to} 23, L is a leaving group, and Pa is a protecting group.) .

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